Large-scale cell culture processes have been developed extensively over the years for the growth of bacteria, yeast and molds, all of which typically possess robust cell walls and/or extra cellular materials thus, are more resilient. The structural resilience of these microbial cells is a key factor contributing to the rapidity of the development of highly-efficient cell culture processes for these types of cells. For example, bacterial cells can be grown in very large volumes of liquid medium using vigorous agitation, culture stirring and gas sparging techniques to achieve good aeration during growth, all the while maintaining viable cultures. Alternatively, bacteria can be grown as a biofilm, however, a growing surface would be required.
In contrast, the techniques to culture cells such as eukaryotic cells, animal cells, mammalian cells and/or tissue are more difficult and complex since these cells are more delicate than microbial cells and have nutrient and oxygen requirements during growth which are more complex and difficult to maintain. Further, animal cells and/or mammalian cells cannot withstand the excessive turbulence and/or shear forces that can be created by an influx of air or gaseous mixtures, such as a mixture containing oxygen, nitrogen and carbon dioxide, that are tolerated more easily by microbial cells. In addition, no animal cells can be directly exposed to gases. Most of the animal cells can only utilize dissolved oxygen in the culture medium. Animal cells and mammalian cells are more likely to be damaged by air and gas influx than are microbial cells and thus, result in increased cell mortality. Bioreactors for larger-scale culturing often have internal moving parts, such as an impeller, which subject the cells to a very high fluid shearing force causing cell damage, sometimes cell death, thus leading to low viability of cultures and as a result reduces protein and/or cell by-product production. Likewise, bioreactors that utilize other types of mechanical parts, harsh air movement, or abrupt fluid movement as a mechanism to achieve cell suspension and/or proper aeration will likely cause damage to cells and hinder cell and tissue growth, which further leads to a decrease in cell by-product production, such a protein.
A primary function of a bioreactor is for research wherein large numbers of cells are grown to refine the minute quantities of an active material, including but not limited to a protein or antibody that are secreted by cells into the growth medium. Another function of a bioreactor is the scale-up laboratory cell culture processes for commercial purposes to mass produce the active proteins made by cells and/or tissues. Because of the need to culture eukaryotic cells and/or prokaryotic cells and/or animal cells and/or mammalian cells in the laboratory in large quantities, bioreactors and culturing devices have become an important tool in research and in the production of cells for producing active proteins and/or antibodies and/or any cell by-products.
Many methods are known in the art for growing cells in culture, both on large and small scales. For smaller-scale cell culturing, many vessels have been developed over the years. Culture dishes, for example, represent one type of culturing vessel. Culture dishes typically consist of a bottom dish, which contains the growth medium, and a removable cover. Although the removable cover provides a convenient access to the culture, cells are often and easily contaminated by microorganisms as a result of repeatingly removing the cover during the culturing process. In fact, contamination is one of the principal challenges to successful cell and tissue culturing techniques.
To overcome contamination with culture dishes, culture flasks were developed. Culture flasks typically have a culture chamber, a small tubular opening located at one end of the flask and a corresponding closure. This design attempts to minimize the exposure of cells to dust, bacteria, yeast and other contaminants. For example, patents teaching culture flasks can be found in U.S. Pat. No. 4,334,028 to Carver and U.S. Pat. No. 4,851,351 to Akamine and U.S. Pat. No. 5,398,837 to Degrassi. Although culture flasks were an improvement over culture dishes, they did not fully remedy the contamination problem. In addition, neither the culture dish nor the culture flask can provide appropriate aeration to cells. Furthermore, the growth surface area available in culture flasks is not adequate, as in culture dishes, thus, placing limits on scaling-up the culturing process using this technology.
Another technology developed for use in cell and tissue culturing were roller bottles. The roller bottle has been widely-used in the art for many years. Although they offer some advantages over dishes and flasks, such as a larger surface area for cell attachment and growth, they are still unable to remedy all of the deficiencies and particularly with scaling up. Collectively, these weaknesses include but are not limited to the large uncontrollable hydrodynamic shear forces associated with a gas headspace and the abundance of turbulent eddies. As a result of the high shear force environment inherent in roller bottles, tissue culturing of larger three-dimensional structures is virtually impossible. Only those cell types that are not damaged by the shear forces and/or are capable of remaining adhered to the wall of the roller bottles can be maintained in culture for an extended period of time. Therefore, long-term maintenance of established cell lines can prove to be difficult with roller bottles due to the constant challenge of the high shear force environment and possible contamination. Examples of patents directed to roller bottles include U.S. Pat. No. 5,527,705 to Mussi et al. and U.S. Pat. No. 4,962,033 to Serkes.
Moreover, although the surface area of roller bottles is greater by comparison to culture flasks and dishes, it is often not considered adequate since the surface for cell adhesion is not necessarily more favorable than the culture flasks and dishes, particularly for scaling up the growth of cell cultures. Some efforts have been made to improve upon the roller bottle by providing a greater amount of surface area per roller bottle. For example, U.S. Pat. No. 5,010,013 to Serkes describes a roller bottle with increased surface area for cell attachment. Serkes relates to the use of corrugated channels added to the interior surface area of the roller bottle to increase capacity for cellular attachment. However, a typical roller bottle provides only a surface area of about 850–1700 cm2 for cultivating cells, a multitude of roller bottles are still required for scaling up production. Although, automation of culturing with a large plurality of roller bottles can save on time and labor investment, these operations are typically costly and limiting.
In addition to the problems of hydrodynamic shear forces and surface-area limitations, a central problem inherent in cell and tissue culturing techniques is attaining and maintaining sufficient oxygenation in the growing culture. It is well-known in the art that prokaryotic cells, eukaryotic cells, including animal cells, mammalian cells, insect cells, yeast and molds all have one major rate-limiting step, oxygen mass transfer.
Oxygen metabolism is essential for metabolic function of most prokaryotic cells and eukaryotic cells with the exception of some fermentative-type metabolisms of various eukaryotic microorganisms, such as yeast. Particularly, with mammalian and animal cell culturing techniques, oxygen flux is especially important during the early stages of rapid cell division. Oxygen utilization per cell is greatest when cells are suspended; requirements for oxygen decrease as the cells aggregate and differentiate. Some mammalian and animal cells are anchorage-dependent, requiring a surface to grow, whereas other mammalian and animal cells are anchorage independent and can be grown in liquid environments regardless of the types of cells. However, these cells all require dissolved oxygen in the medium. Nevertheless, during the later phases of cell culture with both anchorage-dependent and independent cells, as the number of cells per unit volume increases, the bulk oxygen mass transfer requirements once again increases.
Traditionally, at least with anchorage-independent cells, increased requirements for oxygen are accommodated by mechanical stirring methods and the sparging of gases into the culture. However, as discussed, both stirring and the sparging of gases can result in damaging cells, thereby decreasing the viability of the culture and the overall efficiency and productivity of the cell and/or tissue culture. Further, direct sparging of cell and tissue cultures with gas can lead to foam production which, is also detrimental to cell viability.
Some attempts have been made in the art to solve the oxygenation problem during cell culturing. For example, U.S. Pat. No. 5,153,131, issued to Wolf et al. (“Wolf”), relates to a bioreactor vessel without mixing blades. Instead, air travels through an air inlet passageway through a support plate member across a screen and through a flat disk permeable membrane wedged between the two sides of the vessel housing. The oxygen then diffuses across the membrane into the culture chamber due to the concentration gradient between the two sides of the housing.
The Wolf bioreactor, however, presents many disadvantages. Particularly, the rate at which oxygen can diffuse across the disk-shaped membrane is a significant limitation that restricts the size of the culture chamber. Another disadvantage of the flat disk membrane is that it is designed to flex in order to cause mixing within the culture chamber which can result in cell death. The mixing effect is a feature described as being critical for the distribution of air throughout the culture media, however, it will also tend to create shear forces within the chamber, again can be detrimental to cells, consequently providing sufficient gas exchange to sustain the growth of larger cellular structures is a significant and realistic restriction when designing a bioreactor or culture vessel.
An example showing an attempt to overcome the deficiencies thus far described is to make reactors from gas permeable materials. For instance, U.S. Pat. No. 5,702,941, issued to Schwarz et al. (“Schwarz”), entitled “Gas Permeable Bioreactor And Method Of Use” relates to a vessel that is horizontally rotated and the vessel is at least partially composed of gas permeable materials. The gas exchange with the culture medium is intended to occur directly through the gas permeable materials of which the vessel walls are composed.
However, Schwarz discloses that the range of sizes for the vessel is still limited since gas exchange is dependent on the quantity of gas permeable surface area. Schwarz emphasized that as the surface area of the vessel increases, the volume and the amount of culture medium also increases. As such, the preferred dimensions of the vessel described in Schwarz are limited to between one and six inches in diameter while the width is, according to Schwarz, preferably limited to between one-quarter of one inch and one inch. Such size limitations are not suitable for growing three-dimensional cellular aggregates and tissues and/or any scaling up production.
Similarly, U.S. Pat. No. 5,449,617, issued to Falkenberg et al. (“Falkenberg”), entitled “Culture Vessel For Cell Culture” relates to a vessel that is horizontally rotated. The vessel is divided by a dialysis membrane into a cell culturing chamber and a nutrient medium reservoir. Gas permeable materials are used in the vessel walls to enable gas exchange in the cell culturing chamber. However, the vessel is not completely filled with the nutrient medium and a large volume of air is maintained above the fluid medium in both chambers. The Falkenberg vessel, however, is not designed to minimize turbulence within the cell culture chamber but rather, mixing is recited to be an essential step to keep the dialysis membrane wetted. Further, Falkenberg does not contemplate using the vessel to grow cellular aggregates or tissues of any kind.
Others have tried to overcome the oxygenation problem. For example, U.S. Pat. No. 5,766,949, issued to Liau et al. (“Liau”), entitled “Method and Apparatus for Cultivating Anchorage Dependent Monolayer Cells” describes a cell-cultivating system in which the culture medium oscillates up and down with respect to a growth substrate in an attempt to improve the oxygenation of the cells.
Liau, however, presents many disadvantages. One disadvantage of this system is the complexity of Liau's apparatus. The Liau system requires two external storage tanks and a separate growth chamber which holds a series of vertical substrate plates. Multiple peristaltic pumps are required to circulate the growth medium from one storage tank through the culture chamber and then into another storage tank and then back to the first storage tank. Introduction of contaminants is very likely given the complexity and the reliance of the components for the Liau apparatus which are external to the culture chamber, for example, the external tubings, storage tanks, and pumps. Further, sterilization is difficult and laborious due to a relatively large amount of components to the apparatus and the size of apparatus.
Another problem presented by Liau is that the flow of the culture medium through the system would create hydrodynamic shear forces that can easily disrupt and dislodge cells from the substrate plates, thus, reducing the viability of the cells. Furthermore, the vertical substrate plates also discourage cell adhesion since cells that cannot adhere immediately to the plates will simply fall and accumulate at the bottom of the plates and, eventually, most of these cells die. Thus, the culture has a reduced viability, the protein production decreases correspondingly and the system would require continual restarting which is highly inefficient and counterproductive. Moreover, due to the complexity of the system, the harvesting of any secreted protein or cellular product would be cumbersome and time consuming. Lastly, when the growth medium is lowered with respect to the growth substrate plates, the cells become exposed to air, i.e., gaseous environment directly, and thus, may result in cell death. Given the importance of cell and tissue culture technology in biotechnology research, pharmaceutical research, patient care, academic research and in view of the deficiencies, obstacles and limitations exist in the prior art described the present invention overcomes the obstacle and remedies the deficiencies in the prior art by teaching and disclosing a method and an apparatus for cell and tissue culturing that fulfills the long-felt need for a novel method and apparatus to culture cells and tissues that is more reliable, less complex, more efficient, less cumbersome, less expensive, less-labor intensive, capable of increasing cell vitality and producing a higher yield of cellular by-products generated from the cells. The apparatus according to the present invention also reduces contamination, thus increases the longevity of the cell.